1. Field of the Invention
This invention relates generally to the evaluation and care of the extremities and tissues of persons affected by the disease diabetes or other ailments or injuries that may affect the ability to perfuse, oxygenate or heal tissue, particularly to the measurement of changes in tissue oxygenation by natural pressures applied to the foot or other tissues of the body that may lead to ulceration or tissue injury and using this information to offload pressure or provide treatment of injured areas or in areas at high risk and thereby treat or prevent ulceration or other tissue damage.
The present invention is directed to apparati and methods for assessing tissue oxygenation, hydration, oxygen delivery and/or oxygen extraction with hyperspectral imaging and, in particular, tissue oxygenation associated with the foot and other tissues.
2. Description of the Background
Diabetes (or diabetes mellitus) is a chronic disease that affects up to 6% of the US population. When diabetes is present, either the body produces less or no insulin, and/or does not properly use insulin. Insulin is a hormone necessary to maintain blood sugar concentration at normal levels. When insulin is not produced or used correctly by the body, glucose remains in the bloodstream instead of being shuttled into cells for energy production, resulting in high blood glucose, or high “blood sugar” levels.
High blood sugar can manifest its presence through multiple symptoms, including thirst, frequent urination, weight loss, increased hunger, blurred vision, irritability, tingling or numbness in the hands or feet, frequent skin, bladder, or gum infection, wounds that do not heal, and extreme, unexplained fatigue.
If left untreated, diabetes can lead to death, and even diabetics undergoing doctor-supervised treatment suffer an increased death rate compared to the average population. Diabetes is also associated with progressive disease of the microvasculature. Diabetics also face risk of multiple complications during their lifetime arising from the disease. Some of the more serious complications include: heart disease (the leading cause of death in diabetics); stroke (risk of stroke is 2 to 4 times greater for diabetics); high blood pressure (about 73% of diabetics); blindness (diabetic retinopathy causes 12,000 to 24,000 new cases each year and diabetes is the leading cause of new cases of blindness among adults 20-74 years old); kidney disease (diabetes is the leading cause of treated end stage renal disease, accounting for 43% of new cases); nervous system disease (60-70% of diabetics have mild to severe damage, such as impaired sensation of pain in the feet or hands, slowed digestion, and carpal tunnel syndrome); dental disease (almost one-third of diabetics have severe periodontal diseases); pregnancy complications (poorly controlled diabetes before conception and during the first trimester of pregnancy can cause major birth defects in 5-10% of pregnancies and spontaneous abortions in 15-20% of pregnancies); and amputations (more than 60% of non-traumatic lower-limb amputations in the United States occur among diabetics).
Studies of all patients with diabetes under primary care have delivered annual rates of ulcer formation of 5-6%. (Recently reported VA study with an overall annual rate of 6.1% in all patients 40 yrs).1 Stratification into higher risk groups delivers an annual de-novo ulcer formation rate of 33% in patients with a history of amputation, 19% in neuropathic patients with bony deformity and no history of ulcer or amputation and 11% in neuropathic patients with no history of ulcer or amputation.2, 3 
Diabetic neuropathic foot disease is the most common cause of amputation in the United States and arises as a sequella of several of the complications listed above. These complications often stem from the disturbance of the body's metabolism caused by the prolonged high blood sugar. The disturbance includes increased levels of serum cholesterol, triglycerides, and glucosylated hemoglobin, which lead to precipitation of the substances along the small blood vessels (especially capillaries) everywhere in the body, and more so in terminal blood vessels, like those found in the legs and feet. This then leads to damage to or stenosis of the blood vessels, ultimately resulting in a condition termed diabetic microangiopathy, or literally, disease of the capillaries related to diabetes. Longstanding microvascular disease that is widespread may decrease the total capacity of blood circulation within the body, which both directly and indirectly through kidney damage contributes to the high blood pressure condition referenced above. The most dangerous effect of microvascular disease, is occurrence of ischemia (decreased blood supply). This is often manifest in symptoms in the foot and leg, although all tissues may suffer ischemic effects from microvascular disease. This condition can progress with inadequate supply of oxygen and nutrients, eventually producing devitalization and change of texture and color of the foot, often starting with a toe or portion of the forefoot, which can then spread to the rest of the limb. This can take the form of tissue ischemia or frank gangrene.
Diabetic patients also have increased risk of complications associated with their lower extremities, especially the feet, due to nervous system disease, as described above, that can lead to a partial or complete loss of feeling. A healthy person that starts to feel pain when subjected to continuous local pressure may shift their body or make other suitable alterations to automatically lessen the discomfort; however, patients having a sensory loss are deprived of this protection and are therefore common victims of pressure sores and open wounds that can become ulcerated. They also tend to balance themselves differently which can cause progressive alteration in the bony structure of the foot. It is therefore desirable to detect the pressure points or locations of shear stress in the foot to prevent pressure sores and wounds so that a patient who might not be able to recognize existence of a pressure point inducing condition can take curative or preventative measures to eliminate or reduce the condition. More important to just detecting pressure points is to combine this information with the presence of vascular compromise which is the result of a decrease in tissue oxygenation that can be due to a combination of microangiopathy or other influences on adequacy of systemic perfusion to the tissue, large vessel disease due to macrovascular atherosclerosis or obstruction and local factors due to inflammation to an extremity as measured by local tissue oxygenation.
The development of protocols capable of diagnosing potential areas for the development of plantar ulcers would be of great value in decreasing and preventing diabetic foot amputation. Similarly, protocols directed at diagnosing other areas of potential ulceration in diabetic and non-diabetic people, such as sacral ulcers, ulcers on amputation stumps or foot ulcers in athletes would be useful. Special utility would occur in patients with diseases or therapeutic circumstances in which the skin may become fragile such as with scleroderma or other collagen vascular diseases or treatment with steroids.
Diabetic foot lesions are an underlying cause of hospitalization, disability, morbidity, and mortality, especially among elderly people. A protocol for early detection of plantar ulceration would avoid the need for follow-up examinations, supplementary examinations, local wound debridement, orthopedic appliances, and in some critical cases frequent hospitalization, and amputation. Estimates have shown that between 2-6% of diabetic patients will develop a foot ulcer every year,4, 5 and that the attributable cost for an adult male between 40 and 65 years of age is more than $27,000 in 1995 US dollars for the two years after diagnosis of the foot ulcer.4 
Devices are known for indicating to persons having diminished sensation in the foot that their feet are being exposed to excessive stress conditions that could possibly lead to plantar ulcers or worse. Many of these devices include shoes, which detect excess pressure through a force sensor and signal the wearer of the existence that a threshold pressure has been reached. Examples of such devices are described in U.S. Pat. Nos. 5,566,479, 4,610,253, 4,647,918, 5,642,096, and 6,918,883 B2.
Diabetes is a chronic, life-threatening disease for which there is no known cure. It is the fourth leading cause of death in the United States. Over 21 million people in the United States have diabetes and more than 1,000,000 new cases are diagnosed each year. It is estimated that there are at least 194 million people with diabetes worldwide. Type I (or juvenile) diabetes, the most severe form of the disease, comprises 5-10% of diabetes cases and requires daily treatment with insulin to sustain life.
Although medical research experts have not yet found a cure, they have discovered that they can minimize the ravages of diabetes related complications by delineating specific risks, accurately assessing evolving pathologies, and ensuring the rapid institution of effective therapy. This is particularly true in providing appropriate care for the diabetic foot.
The development of an ulcer in the diabetic foot is commonly a result of a break in the barrier between the dermis of the skin and the subcutaneous fat that cushions the foot during ambulation. This rupture can lead to increased pressure on the dermis, resulting in tissue ischemia and eventual death, ultimately manifest in the form of an ulcer.6 
There are a number of factors that weigh heavily in the process of ulceration.7 These factors, such as neuropathy, microcirculatory changes, peripheral vascular disease, obesity and musculoskeletal abnormalities, affect different aspects of the foot, leading to a synergy of effects that greatly increase the risk of ulceration.8 
Neuropathy results in a loss of protective sensation in the foot, exposing patients to undue, sudden or repetitive stress. It can lead to atrophy of the small intrinsic muscles, collapse of the arch, and loss of stability in the metatarsal-phalangeal joints. Neuropathy leads to lack of awareness of damage to the foot as it may be occurring, physical defects and deformities9 which lead to greater physical stresses on the foot. In addition, it can lead to increased risk of cracking and the development of fissures in calluses (a potential entry for bacteria and increased risk of infection).10 
Microcirculatory changes are seen in people with in association with hyperglycemic damage.11 Functional abnormalities occur at several levels. Hyaline basement membrane thickening and capillary leakage may impair diffusion of nutrients. When comparing the microcirculation of the forearm and foot in diabetic patients with and without neuropathy, the endothelium-dependent and endothelium-independent cutaneous vasodilation is lower in the foot.12 On a histologic level, it is well known that diabetes causes a thickening of the endothelial basement membrane which in turn may lead to impaired endothelial cell function.
Peripheral vascular disease (PVD) is “macrovascular disease” caused by atherosclerotic obstruction of large vessels resulting in arterial insufficiency.13 It is more common and more severe in diabetics.14 Like non-diabetics, people with diabetes may develop atherosclerotic disease of large-sized and medium-sized arteries, such as aortoiliac and femoropopliteal atherosclerosis. However, significant atherosclerotic disease of the infrapopliteal segments is particularly common in the diabetic population. The reason for the prevalence of this form of arterial disease in diabetic persons is thought to result from a number of metabolic abnormalities, including high LDL and VLDL levels, elevated plasma von Willebrand factor, inhibition of prostacyclin synthesis, elevated plasma fibrinogen levels, and increased platelet adhesiveness.
Musculoskeletal abnormalities (altered foot mechanics, limited joint mobility, bony deformities) can lead to harmful changes in biomechanics and gait, increasing the pressures associated with various regions of the foot. Alteration or atrophy of fat pads in the foot from increased pressure can lead to skin loss or callus, both of which increase the risk of ulceration by two orders of magnitude.
People with diabetes are more likely to express a combination of the aforementioned factors than non-diabetics, leading the far greater incidence of diabetic foot ulcers in type 1 and type 2 diabetes compared to similar nondiabetics. Clearly, however, foot ulcers can occur in non-diabetics, especially ischemic ulcers seen in patients with peripheral vascular disease and associated with atherosclerosis, hypertension and a history of smoking.
A lower extremity ulcer develops in about 15% of patients with diabetes during their lifetime. Foot pathology associated with vascular disease is a major source of morbidity among diabetics and a leading cause of hospitalization. The infected and/or ischemic diabetic foot ulcer accounts for about 25% of all hospital days among patients with diabetes. Costs of foot disorder diagnosis and management are estimated at over $2 billion annually. Foot ulceration precedes 85% of lower extremity amputations. Proper prevention, evaluation and treatment of diabetic foot disease would clearly improve the quality of life for people with diabetes.
The current market for the diabetes device industry is over $4 billion dollars, and growing 18% annually. This has been primarily in the glucose self-testing area, but demonstrates the large dollars spent annually by patients and the health care system (Medicare and over 60% of other insurers now cover the costs of these devices and supplies.) to take the preventative steps of maintaining better glycemic control to minimize diabetic complications. This demonstrates the huge and growing scope of the overall diabetes market and that this defines the basis of a receptive community of patients and caregivers that will embrace innovative technologies to combat the complications of type 1 and type 2 diabetes such as diabetic foot ulcer.
There is a huge unmet need in prevention, accurate diagnosis and monitoring of therapeutics in diabetic foot disease. Currently the monitoring of pharmacologic therapy is grossly insufficient. Hyperspectral technology will be useful in both drug development and in evaluating clinical progress under a specific pharmacologic therapy. Surgical decision-making will be improved and necessary medical and surgical interventions can be better timed. This will provide huge savings to the health care system. Appropriate pairings of hyperspectral measurements (to deliver a quantitative diagnostic) with therapeutics (both pharmaceuticals and devices) provide diagnostic/therapeutic pairings which can both help the physician select and monitor therapy.
Current solutions are ineffective or incomplete. Diabetic feet are at risk for a wide range of pathologies including infection, ulceration, deep tissue destruction, and/or metabolic complications. Cumulative risks for ulceration include neuropathy, foot-ankle deformity, high planar pressure, poor glucose control, and previous ulceration. Noninvasive techniques now employed in screening for vascular related foot disease have not proven useful in predicting or preventing disease. There is currently no method to assess accurately, rapidly, and noninvasively the predisposition to serious foot complications, to define the real extent of disease or to track the efficacy of therapeutics over time.
Diabetic vascular disease was once thought to involve only the microvasculature. This belief has since been dispelled at both the histologic and surgical levels. It is now possible to perform pedal bypass on the ischemic diabetic leg with improved limb salvage rate and reduction in amputation rates. Although it is possible to have adequate inflow and outflow to the diabetic foot, the microvasculature of the diabetic foot is physiologically altered in terms of flow regulation such that tissue loss can continue to occur.
Functional abnormalities in the microcirculation occur at several levels. Hyaline basement membrane thickening and capillary leakage may impair diffusion of nutrients. When comparing the microcirculation of the forearm and foot in diabetic patients with and without neuropathy, the endothelium-dependent and endothelium-independent cutaneous vasodilatation is lower in the foot.12 On a histologic level, it is well known that diabetes causes a thickening of the endothelial basement membrane which in turn may lead to impaired function of the endothelial cell. Nitric oxide is produced within the endothelial cell and functions to relax smooth muscle cells leading to dilation of the blood vessel. Diabetes, through several molecular mechanisms, functions to decrease the amount of available nitric oxide and thus reduces vasodilatation. The loss of vasodilatation is then thought to lead to early nerve dysfunction through ischemia and nutrient deprivation.15 As neuropathy worsens, the nociceptive C fibers are impaired leading to a loss of the ability to mount a hyperemic response to inflammation.16 This places the foot at risk in terms of infection and the ability to heal minor wounds. Successful revascularization has shown to improve the microcirculation of the skin, but does not completely alter the vasoreactivity or the nociceptive C fiber response.17 This places the revascularized patient still at risk for slow healing of ulcers and infection which may further compromise the foot in spite of adequate inflow.
Although not every diabetic foot disorder can be prevented, it may be possible to effect dramatic reductions in their incidence and morbidity through appropriate prevention and management tools.
Currently available tools for monitoring plantar pressures include pressure sensitive mats (RSscan Labs, UK) and thin in shoe pressure sensitive plates (Tekscan, Boston, Mass.). Other tools are available to measure the contour of the foot including plastic casts and NIR surface scanners (PedAlign, San Diego, Calif.). Specially tailored orthotics are then constructed from information gathered from these measurements that either offload pressure or evenly distribute pressure to the sole of the foot.
A study was recently performed using interferometry for detecting plantar pressure distribution involving a laser light oriented towards a compressed plate.18 This approach involves a pressure plate, which compresses when subjected to a load. The interferogram produced represents the pattern of pressure distribution across the plate. Such approaches as this pose an improvement over the cumbersome, expensive footwear noted above, but this method still suffers from drawbacks, such as ease of use, mass availability, and expense. Further, such methods are only useful for analyzing the bottom or sole of the foot and fails to account for pressure points or locations of shear stress on other parts of the foot. These other methods also do not take into account generalized (systemic), regional or local influences which may decrease perfusion or oxygenation to a given region of the foot.
The effectiveness of these systems to reducing foot ulcerations is still unanswered beyond anecdotal evidence, with groups squaring off between measuring pressure or contour as the important endpoint. It has not been known to measure the spatial distribution of local tissue oxygenation, perfusion oxygen delivery or oxygen extraction while under pressure.
Peripheral Vascular Disease and “Islands of Ischemia”
Another form of ulcer is arterial or ischemic ulcer. These occur in patients with peripheral arterial disease, with or without diabetes. Over 12 million Americans have peripheral arterial disease and the incidence is rising. Ischemic ulcers arise from a lack of perfusion to the tissues adequate to meet the demands of maintaining tissue integrity or of healing a minor injury. The lack of perfusion can be due to blockage of a major vessel, smaller vessels or due to microcirculatory disease. Treatment often requires arterial vascular bypass if this is anatomically feasible. Because of the decrease in perfusion in these ulcers, compression or pressure of any kind is contraindicated.
By reducing flow to the foot, peripheral arterial disease can impede healing; reducing the supply of oxygen and nutrients that tissue requires to maintain the repair process and the viability of the dermal barrier, and significantly amplify the problems associated with diabetic microvascular and neuropathic disease. Each year 343,000 peripheral angiograms, 100,000 peripheral bypasses performed for limb salvage and 135,000 amputations are performed. 82,000 of these amputations are on type 1 and type 2 diabetics. Symptoms and current diagnostic tests are not very sensitive indicators of disease progression or response to pharmacologic therapy.
Rhodes et al. coined the phrase “islands of ischemia” after observing non-healing foot ulcers in diabetic patients despite adequate peripheral bypass.19 In one experiment, a total of fourteen patients were evaluated using Doppler, pulse volume recordings (PVR), and transcutaneous oxygen tensions (TcPO2) in diabetic patients following distal bypass. Group I consisted of eleven patients with no evidence of ulcer following bypass, while Group II consisted of three patients with persistent ulcers despite revascularization. The two groups were compared based on their PVR and TcPO2 results. Both groups were shown to have statistically significant increases in both PVR class and foot TcPO2 (p<0.001). However, despite overall increases in foot TcPO2, the non-healing ulcer group was found to have TcPO2 values less than 20 mm Hg adjacent to the areas of ulceration. This suggests that despite adequate inflow to the extremity with peripheral bypass, “islands of ischemia” exist where inadequate perfusion occurs, thus making the area more susceptible to ulcer formation and inability to heal an ulcer. The etiology of “islands of ischemia” is considered multifactorial and involves abnormal microvascular regulatory mechanisms, histologic changes, and altered neurophysiology.
Venous and Mixed Ulcers
In addition to the diabetic and ischemic ulcers described above, ulcers can also occur primarily associated with venous disease in patients with or without diabetes. About 70% of all leg ulcers are venous ulcers. Venous leg ulcer occurs secondary to underlying venous disease whereby blockage or valve damage leading to valvar insufficiency of the superficial, deep or perforating veins leads to venous hypertension. The ulcer usually presents within the region of the leg just above the ankle. In general, venous ulcers are treated with compression stockings, wraps or bandages. Graduated compression can reduce the elevated pressures in the superficial veins. Compression may also improve the competence of the valves.
Mixed ulcers occur when there is both venous and arterial insufficiency. Generally these present as venous ulcers in someone with some degree of arterial insufficiency. In this circumstance, arterial vascular bypass may also be required. If this is not possible, careful use of compression may be undertaken to help decrease the venous pressure without compromising arterial flow, but this can be difficult to accomplish. Understanding the adequacy of tissue perfusion and oxygenation before undertaking compression therapy is important as is monitoring this during therapy.
Decubitus Ulcers
Sacral and other decubitus ulcers and other forms of pressure sores represent other examples of tissue damage that are to date unable to be prevented or treated in an optimized fashion. They also lead to loss of quality of life, loss of life itself and also represent a huge burden to the health care system. Such ulcers occur in debilitated, hospitalized, paralyzed, malnourished patient groups and in other situations in which pressure is placed on a region of tissue that in some way compromises its viability.
There are also other situations in which abnormalities of skin, vasculature or collagen lead to tissue fragility. This can be associated with a variety of circumstances including malnutrition, cancer, catabolic state, debilitation, steroid use, collagen vascular diseases, and advanced age.
Limp Amputation
Limb amputation is a significant problem due to a variety of causes including trauma, diabetic disease and atherosclerosis. The prevalence of amputation in the United States is approximately 1 million,20 and over 43,000 new major amputations are performed yearly21. The amputee is not only challenged by having the underlying disease or cause of amputation to deal with but also having to learn to use the artificial limb and be beleaguered by the attendant complications that may arise from poor prosthetic fit. This may include recurrent residual limb breakdown predisposing the patient to pain, stump or tissue ulceration or breakdown, osteomyelitis, and sepsis as well as abnormal gait which can occur with improper fit with a secondary result in safety concern, an increase in the energy cost of ambulation and the predisposition to developing osteoarthritis. To date, the evaluation of prosthetic fitting and the addressing of residual limb complications is largely based on limited objective criteria, symptoms and complaints of the amputee and a rather subjective examination of the residual limb, prosthesis, and gait pattern. The implementation of an improved method of assessment of the design of prosthetics would be an advantage. which would encompass both pressure and perfusion or oxygenation data would be an advantage.
Spectroscopy in Medicine
Spectroscopy, like many other analytical techniques, has undergone an evolution in terms of the types of research fields in which it is being utilized. From its early beginnings, it was, and continues to be, a plentiful research field in the hands of physicists. Later, chemists discovered that spectroscopy was a useful tool for the investigation of complex molecular structures. Later still, biologists discovered the usefulness of spectroscopy in the analysis of the structures of biomolecules.22 
Over the last decade, spectroscopy has emerged into medicine. The natural progression of spectroscopy into medicine has paralleled another spectroscopic technique, MRI. The original investigations into problems of medical significance were based on the premise that the biochemistry of a tissue must change before changes in anatomy or morphology, the current standard criteria for many diagnoses, become apparent, and that these biochemical changes will be contained within the spectral signature. Therefore, chemical changes of a disease state should be apparent by spectroscopic analyses prior to any clinical appearance. The progress of research thus far has consistently shown this to be a good premise.22 
Spectra are known to be sensitive to subtle changes in molecular composition and conformation. Spectroscopic analysis of biomolecules is a well established field; and, as any chemist knows, the spectrum of a molecule forms a unique “fingerprint” of that compound. However, this maxim only holds true for pure compounds. Tissues, be they human or animal, are an incredibly complex and highly variable mixture of compounds. The typical spectra obtained from tissue are a weighted average of the spectral features of each of the chemical constituents being sampled within a given sample volume, and as such, these spectra contain information about the biochemical state of the entire sample.
The major obstacle in medical spectroscopy has been sorting out useful diagnostic information from the inter and intra-sample variability. It is not nearly enough to take a spectrum from a healthy piece of tissue and a diseased piece of tissue, compare them, and make valid claims regarding their disease state. It is necessary to take into account the range of disease expressions which occur over a population, as well as the intrinsic variability of tissue spectra during such analysis. This process requires either large, statistically relevant numbers of spectra or a methodology that takes into account the intrinsic inter-sample variability and spatial heterogeneity.
Spectroscopic investigations of medical interest can be roughly divided into three major areas: clinical chemistry, where the goal is to provide a quantitative analysis of blood or other fluid analytes; pathology, which attempts to provide an alternative pathological assessment of a tissue biopsy; and in vivo analyses, where the analysis is done without the need for an invasive procedure. The vision of having a small, inexpensive, portable instrument capable of making a rapid, non-invasive assessment of some relevant medical parameter has provided the driving force behind the application of visible and near-IR spectroscopic techniques to issues of medical interest.
The optical properties of tissue are governed by the bulk scattering properties as well as their absorbance. Variations in tissue or blood analyte composition and/or concentrations will affect visible and near-IR tissue absorbance, while changes in the tissue blood-volume will affect the scattering properties. The interpretation of in-vivo reflectance data is further complicated in that most physical situations which modify tissue absorbance also affect tissue scattering. Visible and near-IR spectroscopic methods have been used for decades in operating theatres in the form of pulse oximeters. These simple systems utilize the different oxyhemoglobin and deoxyhemoglobin absorption bands to determine arterial oxygen saturation.
Skin Spectroscopy
A small portion of visible light shining on the skin of the foot is reflected off the surface. Most of the light passes into the skin through the stratum corneum (˜25 μm thick on the dorsal surface and considerably thicker on the plantar surface of the foot), the epidermis (˜100 μm thick) and into the dermis. The structural features of the dermis (collagen and elastin fibrils, arterial and venous plexus) backscatter the light. This backscattered or re-emitted light maintains the same wavelength spectrum as the incident light, but the intensity is modified by the absorption of skin chromophores.22-25 The intensity modification is directly related to the concentration of chromophores present in the volume of skin investigated. The log of the ratio of the re-emitted to the incident light intensity yields an absorption spectrum of the chromophores.
The primary absorbing chromophores in skin are oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb) (present in the dermis), hemoglobin breakdown products such as bilirubin and methemoglobin, and melanin (present in the epidermis). The spectral properties have been reported.26-28 Hemoglobin has distinct spectral signatures, depending on whether it is oxyHb or deoxyHb. The in-vivo absorption spectra of these compounds have been well-characterized.29 When compared to standard in-vivo absorption spectra, information about the type and also the relative concentration of chromophores in the region of investigation may be quantified.30, 31 
Single point diffuse reflectance (DR) spectroscopy has been used in a variety of studies to investigate the response of the in vivo microvasculature to stimulation. The ratio oxyHb to deoxyHb has been used to derive the oxygen extraction by the tissue, which occurs with metabolism. DR has been used to study oxygen saturation modulation in a variety of tissues and physiologic and pathologic conditions such as pancreatic microcirculation,32 irritant-induced inflammation,33 ischemia-reperfusion injury34 and effect of UV irradiation,35 skin blanch tests.36 Work by Mansfield et al. has shown the utility of DR spectroscopy in the non-subjective diagnosis and monitoring of rheumatoid arthritis37 and basal cell carcinoma.38 
Point spectroscopy of the skin has been shown to be useful for some applications. The understanding derived from previous spectroscopic studies of complex biological systems is essential to accurate design of HT experiments as well as for optimizing the interpretation of imaging data. Understanding the spectroscopic properties of the human body and the physiology of the skin are prerequisites to interpreting HT results.
Hyperspectral Technology
HT or hyperspectral imaging is a method of “imaging” “spectroscopy” that generates a “gradient map” of a region of interest based on local chemical compositions. HT has been used in a wide variety of applications ranging from geological and agricultural to military and industrial, the major airborne applications are in mineral exploration, environmental monitoring and military surveillance.39-42 HT has recently begun to be applied to medicine.43-45 HT for medical applications has been shown to accurately predict viability and survival of tissue deprived of adequate perfusion, and to differentiate diseased (e.g. tumor) and ischemic tissue from normal tissue.
In medicine, spectroscopy is used to monitor metabolic status in a variety of tissues; consider the spectroscopic methods used in pulse oximeters which utilize the different absorption bands oxy- & deoxy-Hb to estimate arterial oxygen saturation. No other method however provides information towards the spatial distribution or heterogeneity of the data. Such spatial information is achieved by HT, where the multi-dimensional (spatial & spectral) data is represented in what is called a “hypercube” (see example in FIG. 2). The spectrum of reflected light is acquired for each pixel in a quadrant and each such spectrum is subjected to standard analysis. From this we create a map of the tissue based on the chemistry of the region of interest.
Tissues have optical signatures that reflect their chemical characteristics, can these can be measured using diffuse reflectance (DR) techniques with an optical probe placed at the site. Tissues have two major optical chromophores of physiological relevance in the visible light spectrum: oxyhemoglobin (OxyHb) and deoxyhemoglobin (DeoxyHb). When measured by hyperspectral technology, these chromophores delineate local oxygen delivery and extraction within the tissue microvasculature. With ischemia, such as in cases of limb ischemia or shock, the spatial composition of OxyHb and DeoxyHb varies across the skin, presenting a mottled appearance. This explains the variability and unreliability seen in tissue oximetry when measured at a single site. Tissue undergoing wound healing also presents varying oxygenation status depending on where the probe is placed relative to the wound. This makes point measurements poor indicators of the wound healing process HT enables the efficient collection of data from over a million points, producing a 2-dimensional map of the state of tissue oxygenation including its spatial variation and thus provides an assessment of “oxygen anatomy.”
Tissue oxygenation mapping is a compelling application of HT. Single point DR spectroscopy has been used to study oxygen saturation in a variety of tissues and physiologic and pathologic conditions such as localized microcirculation, irritant-induced inflammation, ischemia-reperfusion injury, effect of UV irradiation, optical detection of cancer, and peripheral arterial disease. A drawback of single point DR is that it provides no spatial information of tissue oxygenation and for complex systems it is clearly desirable to collect spatial information to monitor local variations, as different regions within the tissue may experience vastly different levels of blood flow, perfusion, and oxygen extraction. This is highly important when assessing either regional blood flow or the area around a wound. Systemic microvascular status, regional blood flow patterns and local physiology all play a role.
Hyperspectral imaging combines the chemical specificity of spectroscopy with the spatial resolution of imaging. In HT light is separated into hundreds of wavelengths using any of a number of possible spectral separators and collected on a charge-coupled device (CCD) in much the same way that a picture is taken by an ordinary camera. In other embodiments, CMOS could be used instead of CCD, or some similar type of sensor. A spectrum of penetrated and reflected light is acquired for each pixel in a region, and each such spectrum can be subjected to standard analysis. This allows the creation of an image representing the chemistry of the region of interest.46 
Hyperspectral Technology (HT), in one guise or another, has become a useful tool for the investigation of spatial heterogeneity in spectral properties in a variety of fields of study ranging from astronomy to medicine. Used for decades in airplane and satellite mounted systems for the mapping of land use and soil types, it has moved in the last five years into a large number of application areas.39-41 Of particular interest here is the use of HT in the fields of biophysics and medicine. The combination of spectroscopic imaging and microscopy has proved very useful in the investigation of the spectral properties of slices of tissue.42, 43 In addition to being useful for the investigation of microscopic structures, HT systems for imaging macroscopic structures have been shown to be useful in the monitoring of the spatial distribution of skin oxygenation.44, 45 HT, however, allows mapping of the regional variations in hemodynamic parameters in response to tissue perfusion.
Changes in the absolute or relative amounts of oxyhemoglobin and deoxyhemoglobin can be measured. Additionally, determining the hemoglobin oxygen saturation (the ratio of oxyhemoglobin divided by the sum of oxyhemoglobin and deoxyhemoglobin) and the total hemoglobin (oxyhemoglobin plus deoxyhemoglobin) is relatively easy given the differing spectra of these two moieties. Unlike single point spectroscopy,23 hyperspectral technology (HT) allows mapping of regional variations in hemodynamic parameters in response to tissue perfusion. Unlike infrared thermography, HT does not map the thermal emission of the tissues. Instead, it relies on the hemoglobin oxygen saturation and other biomarkers of that tissue. One application of HT is in the determination of tissue viability following plastic surgery.47 Tissue which has insufficient oxygenation to remain viable is readily apparent from oxygen saturation maps calculated from near-IR spectral images acquired immediately following surgery; clinical signs of the loss of viability do not become apparent for 6 to 12 hours post-surgery.48 
HT has been studied in a hemorrhagic shock model. An HT system was designed and built for in-vivo use on large animals and human subjects. HT was performed on the ventral surface of the skin in a porcine model. After the image was processed and false colors were applied, light pixels indicated areas of high relative oxygen saturation (O2-sat), whereas dark pixels indicated areas of low O2-sat. It is particularly interesting to note that the mottling seen during hemorrhagic shock features areas of very high tissue oxygenation, alternating with areas of very low tissue oxygenation. The most remarkable finding in these images is the presence of increased regional variability, or “subclinical mottling,” during hemorrhagic shock. As in the plastic surgical model, here HT demonstrated and quantified changes that were not visible to the naked eye. These data indicated early alterations in metabolism. As a more sensitive imaging tool, HT is useful to researchers and clinicians interested in understanding the underlying physiology or monitoring the effects of therapy in their patients.49 
Hyperspectral technology has several features making it a valuable technique for screening and evaluating the foot in diabetes and other peripheral vascular disorders. The technique is noninvasive, rapid, and can be performed during regularly scheduled office visits without the necessity for prior patient preparation. The clinical procedure takes under a minute and requires little more than positioning the patient carefully and taking a pre-programmed series of images at various wavelengths of light with the hyperspectral camera.
Treatment and Prevention of Tissue Breakdown
When tissue breakdown or ulceration is present, therapies are applied to the tissue. In the case of diabetic or ischemic ulcers of the foot, the foot may be offloaded or pressure otherwise relieved from the injured area by bed rest, cut-outs in footwear, total contact casting or other similar treatments. Negative pressure may be applied to assist in healing. In the case of venous ulcers, compression stockings, bandages, wraps or mechanical pumping devices may be applied. Intermittent compression has been used to improve healing of tissue. These therapies have also been applied to prevent tissue breakdown in tissue considered to be at risk for ulceration.
Generally in patients considered to be at risk for diabetic or ischemic ulcer formation, methods are undertaken to evenly distribute pressure to the tissue. In the case of the foot this takes the form of contoured shoe soles, footwear and orthotics and in the case of bed ridden patients air or water beds. However, it is important to understand that in fact to optimize therapy, it should not be uniform pressure that is the goal, but rather applying the least amount of pressure to the areas of tissue most at risk. It would be preferred to identify areas of tissue most at risk and combine this information with contour or pressure mapping data that has been used to apply uniform pressure, to design orthotics or cushions to deliver pressure tailored to the needs of the tissue. In order to prevent diabetic, ischemic, neuropathic or other foot or tissue ulcers patients need more than just uniform pressure relief. Known methods do not solve the mismatch between pressure, perfusion, oxygen delivery and oxygen extraction to meet the demands of the tissue. Foot or other tissue that is poorly perfused or metabolically unstable is more susceptible to the effects of pressure on the region. Therefore, there still remains a need for a method for detecting regions of the foot that are at risk in order to minimize pressure and shear stress especially in regions of poor tissue oxygenation or perfusion.
The applicability of HT in the care of patients with peripheral vascular disease projects into both the clinical setting and operating room. Patients with peripheral vascular disease present with varying degrees of claudication, chronic wounds/ulcers, and gangrene. Non-invasive clinical assessment of these patients is limited. Ankle/brachial indices are limited by both inter-, and intra-observer variability. Ultrasound/laser Doppler only reveals flow within a vessel and not degree of perfusion in the tissue. Transcutaneous oxygen tension can only evaluate a single point at a time. HT bridges the gap between the above modalities and allows real time analysis of tissue perfusion in the entire limb. This will allow the vascular surgeon to evaluate the anatomy specifically and determine which areas of the extremity are non-perfused and which are non-viable. This information will be able to help guide surgical and medical therapy. As an emerging technology, HT shows great promise in the evaluation of tissue perfusion.